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Abstract:

In certain aspects, the invention relates to methods of diagnosing
cervical cancer by using a combination of certain biomarkers such as
hTERT, IGFBP-3, transferrin receptor, beta-catenin, Myc-HPV E6
interaction, HPV E7, and telomere length. In other aspects, the invention
relates to methods of detecting immortalization of cervical cells by
using a combination of certain biomarkers. In yet other aspects, the
invention relates to methods of classifying the grade of a cervical
lesion for diagnostic and prognostic purposes in a female. In further
aspects, the invention relates to methods of treating cervical cancer by
administering a therapeutic agent that targets one or more of these
biomarkers.

Claims:

1. A method of diagnosing or aiding in the diagnosis of cervical cancer in
a female, comprising analyzing the status of at least two biomarkers
selected from the group consisting of: hTERT, IGFBP-3, transferrin
receptor, beta-catenin, Myc-HPV E6 interaction, HPV E7, and telomere
length, in cervical cells of the female.

2. The method of claim 1, wherein, if one of the at least two biomarkers
is hTERT, then the expression level of hTERT is analyzed and increased
expression level of hTERT relative to an appropriate control indicates
that the female has cervical cancer or is at increased risk of developing
cervical cancer.

3. The method of claim 1, wherein, if one of the at least two biomarkers
is IGFBP-3, then the expression level of IGFBP-3 is analyzed and
increased expression level of IGFBP-3 relative to an appropriate control
indicates that the female has cervical cancer or is at increased risk of
developing cervical cancer.

4. The method of claim 1, wherein, if one of the at least two biomarkers
is transferrin receptor, then the expression level of transferrin
receptor is analyzed and increased expression level of transferrin
receptor relative to an appropriate control indicates that the female has
cervical cancer or is at increased risk of developing cervical cancer.

5. The method of claim 1, wherein, if one of the at least two biomarkers
is beta-catenin, then the level of beta-catenin in the cytoplasm and/or
nucleus is analyzed and increased level of beta-catenin in the cytoplasm
and/or nucleus relative to an appropriate control indicates that the
female has cervical cancer or is at increased risk of developing cervical
cancer.

6. The method of claim 1, wherein, if one of the at least two biomarkers
is Myc-HPV E6 interaction, then the association between Myc and HPV E6 is
analyzed and the association between Myc and HPV E6 indicates that the
female has cervical cancer or is at increased risk of developing cervical
cancer.

7. The method of claim 1, wherein, if one of the at least two biomarkers
is HPV E7, then HPV E7 expression is analyzed and the presence of HPV E7
expression indicates that the female has cervical cancer or is at
increased risk of developing cervical cancer.

8. The method of claim 1, wherein, if one of the at least two biomarkers
is telomere length, then the telomere length is analyzed and increased
telomere length relative to an appropriate control indicates that the
female has cervical cancer or is at increased risk of developing cervical
cancer.

9-16. (canceled)

17. A method of detecting immortalization of cervical cells in a female,
comprising analyzing the status of at least two biomarkers selected from
the group consisting of: hTERT, IGFBP-3, transferrin receptor,
beta-catenin, Myc-HPV E6 interaction, HPV E7, and telomere length, in
cervical cells of the female.

18-59. (canceled)

60. A method of classifying the grade of a cervical lesion for diagnostic
and prognostic purpose in a female, comprising: (a) determining the
status of at least two biomarkers in a cervical cell of a female to
provide an individual biomarker diagnostic for cervical lesions, wherein
the at least two biomarkers are selected from the group consisting of:
hTERT, IGFBP-3, transferrin receptor, beta-catenin, Myc-HPV E6
interaction, HPV E7, and telomere length; (b) comparing the status of the
at least two biomarkers from (a) with a biomarker reference panel; and
(c) classifying a cervical lesion for the female by said comparison of
(b).

61. The method of claim 60, wherein, if one of the at least two biomarkers
is hTERT, then the status of the biomarker is the expression level of
hTERT.

62. The method of claim 60, wherein, if one of the at least two biomarkers
is IGFBP-3, then the status of the biomarker is the expression level of
IGFBP-3.

63. The method of claim 60, wherein, if one of the at least two biomarkers
is transferrin receptor, then the status of the biomarker is the
expression level of transferrin receptor.

64. The method of claim 60, wherein, if one of the at least two biomarkers
is beta-catenin, the status of the biomarker is the level of beta-catenin
in the cytoplasm and/or nucleus.

65. The method of claim 60, wherein, if one of the at least two biomarkers
is Myc-HPV E6 interaction, then the status of the biomarker is the
association between Myc and HPV E6.

66. The method of claim 60, wherein, if one of the at least two biomarkers
is HPV E7, then the status of the biomarker is the expression of HPV E7.

67. The method of claim 60, wherein, if one of the at least two biomarkers
is telomere length, then the status of the biomarker is the telomere
length.

68. The method of claim 60, wherein the biomarker reference panel
comprises a constituent panel developed using cervical cancer, high grade
cervical lesion, low grade cervical lesion, and control group
populations.

69-72. (canceled)

Description:

RELATED APPLICATIONS

[0001]This application claims the benefit of the filing date of U.S.
Provisional Application No. 60/488,344, filed Jul. 18, 2003. The entire
teachings of the referenced Provisional Application are incorporated
herein by reference in their entirety.

BACKGROUND

[0003]Cervical cancer is the second most common cancer in women worldwide
with approximately 400,000 new cases being diagnosed each year despite
the existence of screening methods. Infection with human papilloma virus
(HPV) is the cause of almost every case of cervical cancer. Infection
with human papilloma viruses is a common sexually transmitted infection;
more than 50 different viral types are found as human genital infections.
However, only 10-15 types are able to cause cervical cancer and by far
the most common of these are HPV-16 and HPV-18. These viruses encode
transforming oncoproteins E6 and E7 and play a key role in human cervical
cancer.

[0004]There are a number of known methods for diagnosing cervical cancer.
Initial large scale screening relies mainly on cytological screening of
cervical smear samples. Smear samples are taken using routine procedures,
and analyzed for abnormal cell morphology. Samples are then classified in
a number of categories. However, cytological screening is not reliable
and often gives inaccurate results. In individual cases, more invasive
procedures are often necessary to establish a firm diagnosis. Colposcopy
review may be carried out, and in cases where lesions are detected or
suspected, a biopsy may be taken for further more accurate analysis.

[0005]HPV-16 and HPV-18 can be detected in women with undetectable or
minimal cervical abnormality. Thus, cellular factors may therefore
regulate the progression of HPV induced cervical transformation. For
example, it has been suggested that women with a p53 tumor suppressor
protein having an arginine rather than a proline residue at position 72
show an enhanced risk of cervical cancer because the HPV E-6 protein can
cause more efficient degradation of the arginine-containing form of p53,
thereby neutralizing its tumor suppressor function more effectively.

[0006]Clearly, there is a need for additional approaches to diagnosing and
treating cervical cancer which is a significant public health problem.

SUMMARY OF THE INVENTION

[0007]The present invention relates to methods of diagnosing or aiding in
the diagnosis of cervical diseases or conditions, including cervical
cancer, cervical precancer, or immortalization of cervical cells, by
using a panel of biomarkers. The present invention also relates to
methods of treating cervical diseases (e.g., cervical cancer) by
targeting one or more of these biomarkers.

[0008]In one embodiment, the invention provides a method of diagnosing or
aiding in the diagnosis of cervical cancer in a female, who may be of any
age (e.g., child or adult). For example, the female (e.g., girl or woman)
is suspected of having or is known to have cervical cancer (e.g.,
associated with HPV infection). Alternatively, the diagnostic method can
be carried out in any woman, such as during or in conjunction with
routine (regular) healthcare screenings (e.g., periodic physical
examinations). Such method comprises analyzing the status of at least two
of the following biomarkers: human telomerase reverse transcriptase
(hTERT), insulin-like growth factor binding protein 3 (IGFBP-3),
transferrin receptor, beta-catenin, Myc-HPV E6 interaction, HPV E7, and
telomere length, in cervical cells of the female. As used herein, the
status of each biomarker is referred to as follows.

[0009]If the biomarker is hTERT, IGFBP-3, transferrin receptor or HPV E7,
the status to be assessed is the expression level of the biomarker. Thus,
in this method, the expression level of the biomarker is analyzed.
Preferably, the expression level of HPV E7 is analyzed by flow cytometry.
Increased expression level of the biomarker relative to an appropriate
control level (e.g., obtained from a healthy female) indicates that the
female has cervical cancer or is at increased risk of developing cervical
cancer.

[0010]If the biomarker is beta-catenin, the status to be assessed is the
level and localization of beta-catenin in the cytoplasm and/or nucleus.
Thus, in this method, the level and localization of beta-catenin are
analyzed. Increased level of beta-catenin in the cytoplasm and/or nucleus
relative to an appropriate control level (e.g., obtained from a healthy
female) indicates that the female has cervical cancer or is at increased
risk of developing cervical cancer.

[0011]If the biomarker is Myc-HPV E6 interaction, the status to be
assessed is the association between Myc and HPV E6. Thus, in this method,
the association between Myc and HPV E6 is analyzed. Association between
Myc and HPV E6 indicates that the female has cervical cancer or is at
increased risk of developing cervical cancer. Certain aspects of the
invention relate to use of Myc modifications (e.g., phosphorylation) or
mutations in Myc as biomarkers in the methods of the present invention.

[0012]If the biomarker is telomere length, the status to be assessed in
this method is the telomere length. Increased telomere length relative to
an appropriate control length (e.g., obtained from a healthy female)
indicates that the female has cervical cancer or is at increased risk of
developing cervical cancer.

[0013]In another embodiment, the invention provides a method of diagnosing
or aiding in the diagnosis of cervical cancer in a female. Such method
comprises analyzing the status of at least two biomarkers in cervical
cells of the female. One biomarker is Myc-HPV E6 interaction, while a
second biomarker is selected from the group consisting of: hTERT,
IGFBP-3, transferrin receptor, beta-catenin, HPV E7, and telomere length.
The status of each biomarker is described above.

[0014]In still another embodiment, the invention provides a method of
detecting immortalization of cervical cells in a female, who may be of
any age (e.g., child or adult). For example, the female is suspected of
having or is known to have cervical cancer (e.g., associated with HPV
infection). Alternatively, the diagnostic method can be carried out in
any woman, such as during or in conjunction with routine (regular)
healthcare screenings (e.g., periodic physical examinations). Such method
comprises analyzing the status of at least two of the following
biomarkers: hTERT, IGFBP-3, transferrin receptor, beta-catenin, Myc-HPV
E6 interaction, HPV E7, and telomere length, in cervical cells of the
female.

[0015]In a further embodiment, the invention provides a method of
classifying the grade of a cervical lesion for diagnostic and/or
prognostic purposes in a female. Such method comprises: (a) determining
the status of one (or more) biomarker in a cervical cell of a female to
provide an individual biomarker diagnostic for cervical lesions, wherein
the biomarker is selected from the group consisting of: hTERT, IGFBP-3,
transferrin receptor, beta-catenin, Myc-HPV E6 interaction, HPV E7, and
telomere length, and combinations thereof; (b) comparing the status of
the individual biomarker with a biomarker reference panel (e.g., a
reference panel including mean values of the status for the biomarker
constituents of the panel); and (c) classifying a cervical lesion for the
female by said comparison. Preferably, the biomarker reference panel of
the method comprises a constituent panel developed using cervical cancer,
high grade cervical lesion, low grade cervical lesion, and control group
populations.

[0016]In yet another embodiment, the invention provides a method of
treating a female suffering from cervical cancer (e.g., associated with
HPV infection). Such method comprises administering to the female a
therapeutically effective amount of an agent which targets and blocks or
decreases the function (e.g., expression or activity) of one or more of
the biomarkers. In one case, the agent blocks interaction between Myc and
HPV E6. In other cases, the agent blocks or reduces the expression level
of hTERT, IGFBP-3, transferrin receptor, beta-catenin, HPV E6, or HPV E7.
In a particular case, the agent blocks signaling through the beta-catenin
pathway. Exemplary therapeutic agents in such methods include, but are
not limited to, small molecules, polypeptides, antibodies, and nucleic
acids. In specific embodiments, the present invention contemplates the
use of antisense nucleic acids or RNA interference (RNAi) nucleic acids
to block or reduce gene expression of one or more of the above
biomarkers.

[0017]In a further embodiment, the present invention provides a method of
preventing the onset of cervical cancer (e.g., associated with HPV
infection) or reducing the extent to which it occurs in a female. Such
method comprises administering to the female an effective amount of an
agent which targets and blocks or decreases the function (e.g.,
expression or activity) of one or more of the biomarkers. The agent is
effective to prevent the onset of cervical cancer or reduce the extent to
which it occurs. In one case, the agent blocks interaction between Myc
and HPV E6. In other cases, the agent blocks or reduces the expression
level of hTERT, IGFBP-3, transferrin receptor, beta-catenin, HPV E6, or
HPV E7. In a particular case, the agent blocks signaling through the
beta-catenin pathway. Exemplary therapeutic agents in such methods
include, but are not limited to, small molecules, polypeptides,
antibodies, and nucleic acids. In specific embodiments, the present
invention contemplates the use of antisense nucleic acids or RNA
interference (RNAi) nucleic acids to block or reduce gene expression of
one or more of the above biomarkers.

DETAILED DESCRIPTION OF THE INVENTION

[0018]In one embodiment, the invention provides a method of diagnosing or
aiding in the diagnosis of cervical cancer in a female, who may be of any
age (e.g., child or adult). For example, the female is suspected of
having or is known to have cervical cancer (e.g., associated with HPV
infection). Alternatively, the diagnostic method can be carried out in
any woman, such as during or in conjunction with routine (regular)
healthcare screenings (e.g., periodic physical examinations). Such method
comprises analyzing the status of at least two of the following
biomarkers: hTERT, IGFBP-3, transferrin receptor, beta-catenin, Myc-HPV
E6 interaction, HPV E7, and telomere length, in cervical cells of the
female. The status of each biomarker is described above. Discoveries
relating to these biomarkers are described below under the section
"Exemplary Biomarkers for Cervical Cancer."

[0019]In another embodiment, the invention provides a method of diagnosing
or aiding in the diagnosis of cervical cancer in a female. Such method
comprises analyzing the status of at least two biomarkers in cervical
cells of the female. One biomarker is Myc-HPV E6 interaction, while a
second biomarker is selected from the group consisting of hTERT, IGFBP-3,
transferrin receptor, beta-catenin, HPV E7, and telomere length.
Alternatively, one biomarker can be selected from Myc modifications
(e.g., phosphorylation) and mutations in Myc.

[0020]In still another embodiment, the invention provides a method of
detecting immortalization of cervical cells in a female. Such method
comprises analyzing the status of at least two of the following
biomarkers: hTERT, IGFBP-3, transferrin receptor, beta-catenin, Myc-HPV
E6 interaction, HPV E7, and telomere length, in cervical cells of the
female.

[0021]In yet another embodiment, the invention provides a method of
treating a female suffering from cervical cancer (e.g., associated with
HPV infection), and a method of preventing the onset of cervical cancer
or reducing the extent to which it occurs in a female. Such methods
comprise administering to the female a therapeutically effective amount
of an agent which targets and blocks or decreases the function (e.g.,
expression or activity) of one of the biomarkers. In one case, the agent
blocks interaction between Myc and HPV E6. In other cases, the agent
blocks or reduces the level of expression of hTERT, IGFBP-3, transferrin
receptor or beta-catenin. In a particular case, the agent blocks
signaling through the beta-catenin pathway. Exemplary therapeutic agents
in such methods include, but are not limited to, small molecules,
polypeptides, antibodies, and nucleic acids. In specific embodiments, the
present invention contemplates the use of antisense nucleic acids or RNA
interference (RNAi) nucleic acids to block or reduce gene expression of
one or more of the above biomarkers.

Exemplary Biomarkers for Cervical Cancer

[0022]The present invention contemplates use of certain biomarkers in
diagnosing and treating cervical cancer. In specific embodiments, these
biomarkers can be used in detecting immortalization of cervical cells.
Examples of biomarkers for the present invention include, but are not
limited to, hTERT, IGFBP-3, transferrin receptor, beta-catenin, Myc
(e.g., Myc-HPV E6 interaction, a myc modification or a mutation in Myc),
HPV E7, and telomere length.

1) hTERT

[0023]Telomerase activity is detected in more than 90% of immortalized and
cancer cells but absent in most normal somatic cells (Kim et al., 1994,
Science, 266:2011-2015; Meyerson et al., 1997, Cell, 90:785-795),
suggesting that telomerase activation is an important event during the
process of immortalization and malignant transformation. Telomerase
activity is closely associated with the expression of the telomerase
catalytic subunit, hTERT. The expression of hTERT RNA is detected at high
levels in tumor tissues and tumor-derived cell lines but not in normal
adjacent tissues or primary cells (Ramakrishnan et al., 1998, Cancer
Res., 58:622-625; Takakura et al., 1998, Cancer Res., 58:1558-1561). It
is suggested that hTERT is the rate-limiting determinant of enzymatic
activity of human telomerase and that upregulation of hTERT might be a
critical event in the development of human cancers.

[0024]Over-expression of hTERT is responsible for the increase in cellular
telomerase activity (see, e.g., Veldman et al., 2001, J Virol.,
75:4467-72; Yuan et al., 2002, J Virol., 76:10685-91). This activity is
essential for cell immortalization and for cancer progression. More
importantly, hTERT expression exhibits a very large increase during
immortalization, independent of HPV gene expression (Baege et at, 2002,
Am J Pathol., 160:1251-7). Thus, this increased expression of hTERT can
be used to confirm or detect immortalization, an important step in the
progression of HPV-infected cells to the immortal state, and can be used
to differentiate between cervical cells which are simply infected but not
immortalized by HPV and those cells that are infected and immortalized by
HPV. This marker alone, or in combination with other markers described
herein, provides a diagnostic tool for detecting cervical cancer cells,
such as those cells which have progressed from the HPV-infected state to
the immortal state. Further, hTERT can be a therapeutic target for
cervical cancer.

2) IGFBP-3

[0025]Insulin-like growth factor binding protein 3 (IGFBP-3), the most
abundant IGFBP in human serum, is synthesized mainly by hepatic Kupffer
cells and binds over 90% of circulating IGF, resulting in a prolonged
half-life of IGF (Baxter et al., 1989, Prog. Growth Factor Res.,
1:49-68). IGFBP-3 is also produced locally by a variety of normal and
tumor cells. The biological functions of IGFBP-3, aside from being the
major binding protein for IGF-1, are complex and remain poorly
understood. IGFBP-3 modifies the interaction of IGF-1 with its receptor
(Kelly et al., 1996, Int. J. Biochem. Cell Biol., 28:619-637), and
modulates IGF-1 activity by binding to the extracellular matrix and cell
surfaces, possibly to yet identified receptors (Baxter et al., 2000, Am.
J. Physiol. Endocrinol. Metab., 278:967-976). Furthermore, it has been
suggested that IGFBP-3 may signal independently of IGF and that it can be
translocated to the nucleus where it interacts with nuclear components,
which remain to be identified (Baxter et al., 2001, Mol. Pathol.,
54:145-148).

[0026]IGFBP-3 is over-expressed in immortalized cervical cells. Similar to
hTERT, IGFBP-3 shows a large increase (˜500 fold) in expression
during the process of cell immortalization (Berger et al., 2002, Am. J.
Pathol., 161:603-610). In addition,

[0027]IGFBP-3 can be a positive regulator of IGF-1 signaling and appears
to have an important role in sensitizing cervical cells to IGF-1. Thus,
IGFBP-3 can augment the growth of cervical cells and may have a critical
role in cervical cancer. In particular, IGFBP-3 alone, or in combination
with other markers described herein, can potentially be used to detect
cervical cancer cells. Further, IGFBP-3 can be a therapeutic target for
cervical cancer.

3) Transferrin Receptor

[0028]Transferrin receptor is present on almost all mammalian cells.
Transferrin receptor binds the major serum iron-transport protein,
transferrin, and mediates cellular iron uptake by receptor-mediated
endocytosis.

[0029]Cervical cancer cells over-express transferrin receptor.
Over-expression of transferrin receptor can be used, alone or in
combination with other markers as described herein, as a diagnostic
marker for detecting cervical cancer cells. Further, transferrin receptor
can be a therapeutic target for cervical cancer.

4) Beta-catenin

[0030]β-catenin protein functions in two independent processes:
cell-cell adhesion and signal transduction (Peifer, 1997, Science,
275:1752-3). In the adherence junctions, it binds to the cytoplasmic tail
of E-cadherin and mediates the interaction between the adherence
junctions and actin microfilaments with α-catenin. In cells,
β-catenin is localized mostly in such adherent junctions, and the
free cytoplasmic β-catenin level is very low. Elevation of the free
β-catenin level in the cytoplasm can be caused by mutation of
β-catenin itself (Rubinfeld et al., 1997, 275:1790-2).

[0031]The conversion of immortal genital cells to the tumorigenic
phenotype is accompanied by the increased expression of β-catenin in
the cytoplasm and/or nucleus. This provides an important understanding of
the progression of cervical cancer. β-catenin has been observed to
be expressed in cervical cancer but its gene is not mutated. In addition,
Applicants' findings help understand the mechanism for the increased
expression of β-catenin in the cytoplasm and/or nucleus. Applicants
also identified the β-catenin pathway that contributes to the
conversion to cervical cancer. Therefore, the β-catenin pathway
(e.g., expression and/or localization of β-catenin in the cytoplasm
and/or nucleus) can be used alone, or in combination with other markers
as described herein, as diagnostic biomarkers for detecting cervical
cancer cells. Further, the β-catenin pathway can potentially offer
several therapeutic targets (e.g., β-catenin) for cervical cancer.

5) Myc

[0032]Myc protein is a critical regulator of epithelial cell growth and
differentiation. c-Myc, as a transcription factor, can promote cell
proliferation by regulating the expression of numerous target genes.
Applicants recently discovered that Myc protein is a target of the HPV E6
protein in cervical cancer. Specifically, Myc associates with HPV E6
protein and cooperatively activates the hTERT promoter (Veldman et al.,
2003, Proc Natl Acad Sci U S A., 100:8211-6). Thus, Applicants suggest
that Myc/E6 interaction can be used alone, or in combination with other
markers as described herein, for detecting cervical cancer cells. In
addition, Applicants suggest that therapeutic approaches for cervical
cancer can be designed by interfering with the Myc/E6 interaction.

[0033]Optionally, Myc modifications and/or mutations can be used as
possible diagnostic markers for detecting cervical cancer. For example,
activities of Myc protein may be regulated by post-translational
modifications which include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
To illustrate, there are two major phosphorylation sites in the
N-terminal transactivation domain of Myc (Thr-58 and Ser-62) that
regulate transcriptional and transforming activities of Myc (Henriksson,
et al., 1993, Oncogene 8:3199-3209; Blackwood et al., 1991, Science
251:1211-1217). Thus, certain modifications of Myc (e.g.,
phosphorylation) may be associated with cancers such as cervical cancer.
Further, it is possible to screen for and identify mutations in Myc which
alter Myc activities. Thus, certain mutations in Myc may also be
associated with cancers such as cervical cancer. Applicants propose that
Myc modifications and/or mutations may have an important role in tumor
progression and in cancer diagnosis.

6) HPV E7 Expression

[0034]Two oncoproteins, E6 and E7, are encoded by the high-risk HPVs. Both
HPV E6 and HPV E7 can form specific complexes with tumor suppressor gene
products. The HPV E7 protein binds to the retinoblastoma tumor suppressor
gene product (pRB). The HPV E6 protein can associate with the p53 tumor
suppressor protein. The functional inactivation of pRB and p53 by the HPV
oncoproteins E7 and E6, respectively, are likely to be important steps in
cervical carcinogenesis.

[0035]Applicants have recently shown that it is possible to detect
expression of HPV E7 protein in cervical cancer cells by flow cytometry.
This technique could be used to rapidly identify HPV-infected cells. In
combination with other markers described herein, this can be the basis
for a rapid assay for cervical cancer cells. For example, HPV E7 gene
expression can be detected by performing polymerase chain reactions (PCR)
inside of intact cells. Measurement of genetic parameters and observation
of genetic properties while maintaining the integrity of the DNA or RNA
in a cell is then accomplished by passing a suspension of cells through a
flow cytometer wherein the properties and parameters can be measured on a
cell by cell basis. Specifically, cells are first fixed by suspension in
a solution comprising ultrapure formaldehyde and then removed from the
solution. A polymerase is then added into the cells to amplify specific
genetic material (e.g., E7). Finally, the amplified genetic material in
individual cells is rapidly detected by a flow cytometer.

[0036]A flow cytometer is an instrument that will measure fluorescence of
individual cells as they pass in single file through a light source
(usually a laser beam). Antibodies labeled with fluorescent dyes directed
against cell antigens, fluorescent dyes that label specific substrates in
the cell and fluorochromes that are sensitive to ions have all been used
to label specific cell populations or molecules within cells for
identification and evaluation of function. Flow cytometry can also be
used to sort cells.

[0037]As cells labeled with a fluorochrome attached in some way to the
desired component pass through the laser beam, the fluorochrome is
excited. The emission is detected orthogonally (perpendicular) to the
laser beam as the light passes through a focusing lens system and
spectral filters to selectively detect the desired wavelength. The light
is then detected by a photomultiplier tube that integrates all the
fluorescence that passes through the color bandpass filter. Nonspecific
cellular fluorescence called autofluorescence appears yellow to the eye
but there is a significant green component to it and this component is
passed along with the green fluorescein fluorescence from fluorescein
through the bandpass filter. Thus, the flow cytometer detects both the
autofluorescence and the specific fluorescence from the component that is
stained with fluorescein. If the fluorescence of the stained component is
too low, it will not be resolved from the autofluorescence. A method to
amplify the fluorescence of the desired component above the
autofluorescence and other nonspecific fluorescence has been developed.

7) Telomere Length

[0038]Telomeres containing noncoding DNA repeats at the end of the
chromosomes are essential for chromosomal stability and are implicated in
regulating the replication and senescence of cells. The gradual loss of
telomere repeats in cells has been linked to aging and tumor development.
As described above, Applicants have evidence that hTERT is over-expressed
during immortalization. Applicants also found that increased telomere
length results from this increased telomerase activity. It is therefore
possible to identify immortalized cells with fluorescent probes for
telomere length, and may be used alone, or in combination with other
markers described herein, as a diagnostic approach for cervical cancer.

[0039]Procedures and methods for measuring telomere length are known in
the art and can be used in this invention. For detection of telomeric
length, one may study just a particular cell type, all cells in a tissue
(where various cells may be present), or subsets of cell types, and the
like. The preparation of the DNA having such telomeres may be varied,
depending upon how the telomeric length is to be determined. At least
three methods for measuring the length of telomere repeats have been
described: Southern blot analysis and quantitative fluorescence in situ
hybridization using either digital fluorescence microscopy (Q-FISH) or
flow cytometry (flow-FISH). See, e.g., Allshire et al., 1988, Nature,
332:656-659; de Lange et al., 1990, Mol. Cell Biol. 10:518-527; Rufer et
al., 1998, Nature Biotech, 16: 743-747; and Poon et al., 1999, Cytometry,
36:267-278. Methods for measuring telomere length are also described in,
for example, U.S. Pat. Nos. 6,368,789 and 6,551,774.

[0040]For example, Southern blot analysis is a multi-step method which
entails: (a) cleaving purified DNA with restriction enzymes; (b)
separating the DNA fragments by size on an agarose gel; (c) denaturing
and transferring the DNA fragments to a membrane; (d) hybridizing the
telomere with a radioactive telomere probe; (e) removing the unhybridized
probe by washing the membrane; and (f) analyzing the data by
autoradiography and image analysis (see, e.g., Allshire et al., 1988,
Nature, 332:656-659; de Lange et al., 1990, Mol. Cell Biol. 10:518-527).
In addition, several alternative methods have been described in recent
years. Some, such as pulsed-field electrophoresis, slot blots, and
centromere-to-telomere ratio measurements are essentially improvements to
the Southern blot technique.

[0041]However, other methods, such as fluorescent in situ hybridization on
metaphase chromosome spreads and flow cytometry-based fluorescent in situ
hybridization, represent a new technical approach to the problem (see,
e.g., Rufer et al., 1998, Nature Biotech, 16: 743-747; Poon et al., 1999,
Cytometry, 36:267-278). For example, in the flow-FISH technique, a
fluorescein isothiocyanate (FITC)-labeled telomere-specific peptide
nucleic acid (PNA) probe is hybridized in a quantitative way to telomere
repeats, followed by telomere fluorescence measurements on individual
cells by flow cytometry.

Methods of Diagnosis

[0042]In certain embodiments, the present invention provides methods of
diagnosing or aiding in the diagnosis of cervical cancer in a female. In
certain embodiments, the present invention relates to methods of
detecting immortalization of cervical cells in a female as it is known
that immortalized cervical cells have striking parallels to high-grade
cervical lesions. For example, when HPV-16- or HPV-18-immortalized
cervical cells are grown in raft cultures, they form structures similar
to high-grade cervical lesions, characterized by a lack of stratification
and differentiation, an expansion of basal-type cells throughout the
epithelium, and cellular disorganization and nuclear atypia (see, e.g.,
Rader et at, 1990, Oncogene, 5:571-576; Pecoraro et al., 1991, Am J
Pathol, 138:1-8). These methods of diagnosis comprise detecting the
status of a biomarker selected from hTERT, IGFBP-3, transferrin receptor,
beta-catenin, Myc-HPV E6 interaction, HPV E7, and telomere length, or
combinations thereof, in cervical cells of the female.

[0043]In certain embodiments, the present invention provides methods of
classifying the grade of a cervical lesion for diagnostic and/or
prognostic purposes in a female. For example, such method comprises the
following steps: (a) determining the status of one or more biomarkers in
a cervical cell of a female to provide individual biomarkers diagnostic
for cervical lesions, wherein the biomarkers are selected from the group
consisting of: hTERT, IGFBP-3, transferrin receptor, beta-catenin,
Myc-HPV E6 interaction, HPV E7, and telomere length; (b) comparing the
status of the biomarkers with a biomarker reference panel (e.g., a
reference panel including mean values of the status for the biomarker
constituents of the panel); and (c) classifying a cervical lesion for the
female by said comparison. Preferably, the status of the biomarker is
significantly higher for a high grade cervical lesion (e.g., cervical
cancer) than for a low grade cervical lesion (e.g., cervical precancer).

[0044]For example, the method of the invention can be used for classifying
(or categorizing) female subjects in one of several diagnostic groups. In
increasing order of severity, these groups include "negative," "low grade
squamous intraepithelial lesions (LGSIL: HPV-CIN1)," "high grade squamous
intraepithelial lesions (HGSIL: CIN2-CIN3)," and "cervical cancer."
Applicant has established that presence or absence of a biomarker gene
product (e.g., mRNA or protein), level of expression of a biomarker gene
product, subcellular (e.g., cytoplasm or nucleus) localization or level
of a biomarker gene product, interaction of a biomarker protein with its
associated protein, or telomere length in a cell sample (e.g., cervical
cells), may be used to give an accurate predictor of the final diagnostic
group of a female from which the sample is taken. For example, presence
of HPV E7 in a sample may be indicative of increased susceptibility to
cervical cancer. As another example, mean values of a biomarker mRNA or
protein in a sample can show statistical differences between samples from
patients in each of the final diagnostic groups. To illustrate, a sample
from an HGSIL patient may have significantly higher levels of hTERT,
IGFBP-3, and/or transferrin receptor than one from an LGSIL patient and
one from a normal (healthy) female. The phrase "significantly higher" as
described herein is well within the knowledge of a skilled artisan, and
will be determined empirically with reference to the particular
biomarker. For example, the phrase "significantly higher" is relative to
an appropriate control level (e.g., a level determined in samples from
healthy females).

[0045]Optionally, the biomarker reference panel of the method comprises a
constituent panel developed using cervical cancer, high grade cervical
lesion, low grade cervical lesion, and control group populations. The
reference panel includes one or more biomarkers identified as having
diagnostic value, such as the biomarkers described in this application as
well as other biomarkers for cervical cancer (e.g., E6 or E7 proteins).
Optionally, each referenced biomarker constituent of the panel can have a
range of values that correspond to various diagnostic groups.

[0046]The term "cervical cells" as used herein, refers to cell samples
(e.g., primarily a collection of cells) from the cervix of a patient (a
subject or an individual, preferably a female). One method of obtaining
cells is through non-invasive means, which is defined herein as obtained
without the puncturing of a patient. Examples of non-invasive means are,
for example, cell samples obtained from cervical or other cell surface
scrape. Patient cells can also be obtained by other means including, for
example, needle biopsy or tissue biopsy.

[0047]The cervical cells used in the present invention can be preserved in
a collection medium which allows for a combination of two or more assays
of different characteristics related to a cell state of interest. As used
herein, the assay or assays refer to a section or measurement of specific
characteristics, the results of which may be combined with other such
measurements of other characteristics to provide an overall assessment of
a cell suspected of being affected by one or more diseases or conditions.
These assays may include, for example, a combination of morphological
analysis and quantitation of a particular RNA or DNA or protein whose
levels provide a specific indication of the presence or progression of a
disease. Alternatively, for example, the collection medium can be used to
combine an assay identifying the morphology of cells in a cell sample
with one or more assays identifying the HPV type involved, and, for
example, identifying whether the HPV type identified is a high risk or
low risk HPV type for the development of HPV-induced cell transformation
and cancer.

[0048]Cervical cell samples for use in the present invention can be
collected and stored in liquid medium. Examples of useful cell collection
media are STM (Digene), PreservCyt® (Cytyc), and CytoRich®
(Autocyte). These media (PreservCyt® and CytoRich®) were
developed for the collection of cytological samples but can be adapted,
for use with molecular assays.

[0049]Cervical cell samples for use in the method of the present invention
can be fixed or processed in any manner consistent with the assays to be
performed. For example, both cytological and molecular assays can be
performed using cells fixed on a solid substrate such as, for example, a
slide. The requirements of the assays to be performed will generally
identify the sample processing to be used.

[0050]In further embodiments, the present invention provides a kit
suitable for use in the present diagnostic methods. For example, those
methods can be conveniently performed using kits that include one or more
of the materials needed for the method, such as reagents and sample
collection and handling materials. For example, kits can include cell
collection medium, sample preserving reagents, reagents for specific
detection of DNA and/or expression products (RNA or proteins) of one or
more of the biomarkers (e.g., hTERT, IGFBP-3, transferrin receptor,
beta-catenin, Myc-HPV E6 interaction, HPV E7, and telomere length) and
sample handling containers. Useful reagents for detection of gene
expression of certain biomarkers are nucleic acid probes for those genes.
A kit may also contain control samples or reagents, or reagents and
materials for performing other assays to be combined with the disclosed
assay. In addition, the kits can contain reagents for the separation of
RNA and/or DNA from other cellular components.

[0051]The present invention can be performed using devices adapted to the
method. Numerous devices for performing similar assays are known and in
use and can be adapted for use with the disclosed assays and method. For
example, devices are known for automating all or a part of sample assays
and sample handling in assays.

[0052]In certain embodiments, diagnostic methods of the present invention
can include the combination with any other assays for assessing a disease
or state of cells in a cell sample. For example, the subject diagnostic
methods can be combined with cytological assays, histological assays,
determination of the HPV type, determination of the level of HPV, assays
detecting other cellular markers such as oncoproteins or tumor
suppressors, or any combinations of these assays. Such assays are known
and are used for the diagnosis of HPV infection or cervical diseases
(e.g., cervical cancer) and assessment of the stage of the cervical
disease. Results from the subject diagnostic methods and one or more
additional assays can be combined to increase the reliability of any
assessment, prognosis, diagnosis, or monitoring of cervical diseases.
Where multiple assays point in the same prognostic or diagnostic
direction, the reliability of the assessment is increased. Combined
assays can be performed in any order and in any temporal relationship.
For example, various assays can be performed in parallel or
simultaneously. Such assays can be performed in any manner such as on the
same apparatus by the same person, with different apparatus, or in the
same or different locations.

[0053]For example, cytological assays for use in assessing the stage of
HPV-based diseases (e.g., cervical cancer) are known and can be combined
with the disclosed method. The well established Pap smear and Hematoxylin
& Eosin stains (H&E) are preferred examples. The use and analysis of Pap
smears and H&E stains are well-known in the art.

[0054]Methods of the present invention involve noninvasive procedures
which are suitable for large scale screening of patients and are more
accurate than conventional cytological screening. Further, since the
subject methods can be used for predicting the final diagnostic group of
a patient by classifying the grade of a cervical lesion, it is possible
to select the treatment most appropriate for that patient. For example,
an LGSIL patient can simply be closely monitored rather than subjected
unnecessarily to the more harsh aggressive treatment appropriate for a
HGSIL patient.

Methods of Treatment

[0055]In certain embodiments, the present invention provides methods of
treating a female suffering from cervical cancer, as well as methods of
preventing the onset of cervical cancer in a female. As used herein, a
therapeutic (therapeutic agent or therapeutic compound) that "prevents" a
disorder or condition refers to a compound that, in a statistical sample,
reduces the occurrence of the disorder or condition in the treated sample
relative to an untreated control sample, or delays the onset or reduces
the severity of one or more symptoms of the disorder or condition
relative to the untreated control sample. Thus, prevention of cervical
cancer includes, for example, reducing the number of detectable cancerous
growths in a population of patients receiving a prophylactic treatment
relative to an untreated control population, and/or delaying the
appearance of detectable cancerous growths in a treated population versus
an untreated control population, e.g., by a statistically and/or
clinically significant amount. The term "treating" as used herein
includes prophylaxis of the named condition or amelioration or
elimination of the condition once it has been established.

[0056]In certain embodiments, methods of the invention comprise
administering to the female a therapeutically effective amount of a
therapeutic agent which targets one or more of the biomarkers as
described above (e.g., hTERT, IGFBP-3, transferrin receptor,
beta-catenin, and Myc-HPV E6 interaction). To illustrate, a therapeutic
agent of the invention can block or reduce the level of expression of
hTERT, IGFBP-3, transferrin receptor or beta-catenin. Alternatively, a
therapeutic agent of the invention can block interaction between Myc and
HPV E6. The therapeutic agents of the present invention include, but are
not limited to, a polypeptide, an antibody, a small organic molecule, a
peptidomimetic, and a nucleic acid.

[0057]In certain aspects, the therapeutic agents may include a polypeptide
and an antibody. Such therapeutic agents can, for example, prevent the
interaction between Myc or HPV E6, or block signaling through the
beta-catenin pathway. Antibodies may be polyclonal or monoclonal; intact
or truncated, e.g., F(ab')2, Fab, Fv; xenogeneic, allogeneic, syngeneic,
or modified forms thereof, e.g., humanized, chimeric, etc.

[0058]In certain aspects, the therapeutic agents of the present invention
include a nucleic acid. In one embodiment, the invention relates to the
use of antisense nucleic acid to decrease expression of one or more of
the biomarkers (e.g., hTERT, IGFBP-3, transferrin receptor or
beta-catenin). Such an antisense nucleic acid can be delivered, for
example, as an expression plasmid which, when transcribed in the cell,
produces RNA which is complementary to at least a unique portion of the
cellular mRNA which encodes a biomarker polypeptide (e.g., hTERT,
IGFBP-3, transferrin receptor or beta-catenin). Alternatively, the
construct is an oligonucleotide which is generated ex vivo and which,
when introduced into the cell causes inhibition of expression by
hybridizing with the mRNA and/or genomic sequences encoding a biomarker
polypeptide. Such oligonucleotide probes are optionally modified
oligonucleotide which are resistant to endogenous nucleases (e.g.,
exonucleases and/or endonucleases), and are therefore stable in vivo.
Exemplary nucleic acid molecules for use as antisense oligonucleotides
are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA
(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).
Additionally, general approaches to constructing oligomers useful in
nucleic acid therapy have been reviewed, for example, by van der Krol et
al., (1988) Biotechniques 6:958-976; and Stein et al., (1988) Cancer Res
48:2659-2668.

[0059]In another embodiment, the invention relates to the use of RNA
interference (RNAi) to effect knockdown of one or more of the biomarker
genes (e.g., hTERT, IGFBP-3, transferrin receptor or beta-catenin). RNAi
constructs comprise double stranded RNA that can specifically block
expression of a target gene. "RNA interference" or "RNAi" is a term
initially applied to a phenomenon observed in plants and worms where
double-stranded RNA (dsRNA) blocks gene expression in a specific and
post-transcriptional manner. RNAi provides a useful method of inhibiting
gene expression in vitro or in vivo. RNAi constructs can comprise either
long stretches of dsRNA identical or substantially identical to the
target nucleic acid sequence or short stretches of dsRNA identical to
substantially identical to only a region of the target nucleic acid
sequence.

[0060]Optionally, the RNAi constructs contain a nucleotide sequence that
hybridizes under physiologic conditions of the cell to the nucleotide
sequence of at least a portion of the mRNA transcript for the gene to be
inhibited (e.g., the "target" gene). The double-stranded RNA need only be
sufficiently similar to natural RNA that it has the ability to mediate
RNAi. Thus, the invention has the advantage of being able to tolerate
sequence variations that might be expected due to genetic mutation,
strain polymorphism or evolutionary divergence. The number of tolerated
nucleotide mismatches between the target sequence and the RNAi construct
sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in
20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA
duplex are most critical and may essentially abolish cleavage of the
target RNA. In contrast, nucleotides at the 3' end of the siRNA strand
that is complementary to the target RNA do not significantly contribute
to specificity of the target recognition. Sequence identity may be
optimized by sequence comparison and alignment algorithms known in the
art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press,
1991, and references cited therein) and calculating the percent
difference between the nucleotide sequences by, for example, the
Smith-Waterman algorithm as implemented in the BESTFIT software program
using default parameters (e.g., University of Wisconsin Genetic Computing
Group). Greater than 90% sequence identity, or even 100% sequence
identity, between the inhibitory RNA and the portion of the target gene
is preferred. Alternatively, the duplex region of the RNA may be defined
functionally as a nucleotide sequence that is capable of hybridizing with
a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES
pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16
hours; followed by washing).

[0061]The double-stranded structure may be formed by a single
self-complementary RNA strand or two complementary RNA strands. RNA
duplex formation may be initiated either inside or outside the cell. The
RNA may be introduced in an amount which allows delivery of at least one
copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000
copies per cell) of double-stranded material may yield more effective
inhibition, while lower doses may also be useful for specific
applications. Inhibition is sequence-specific in that nucleotide
sequences corresponding to the duplex region of the RNA are targeted for
genetic inhibition.

[0062]The subject RNAi constructs can be "small interfering RNAs" or
"siRNAs." These nucleic acids are around 19-30 nucleotides in length, and
even more preferably 21-23 nucleotides in length. The siRNAs are
understood to recruit nuclease complexes and guide the complexes to the
target mRNA by pairing to the specific sequences. As a result, the target
mRNA is degraded by the nucleases in the protein complex. In a particular
embodiment, the 21-23 nucleotides siRNA molecules comprise a 3' hydroxyl
group. In certain embodiments, the siRNA constructs can be generated by
processing of longer double-stranded RNAs, for example, in the presence
of the enzyme dicer. The combination is maintained under conditions in
which the dsRNA is processed to RNA molecules of about 21 to about 23
nucleotides. The siRNA molecules can be purified using a number of
techniques known to those of skill in the art. For example, gel
electrophoresis can be used to purify siRNAs. Alternatively,
non-denaturing methods, such as non-denaturing column chromatography, can
be used to purify the siRNA. In addition, chromatography (e.g., size
exclusion chromatography), glycerol gradient centrifugation, affinity
purification with antibody can be used to purify siRNAs.

[0063]Production of RNAi constructs can be carried out by chemical
synthetic methods or by recombinant nucleic acid techniques. Endogenous
RNA polymerase of the treated cell may mediate transcription in vivo, or
cloned RNA polymerase can be used for transcription in vitro. The RNAi
constructs may include modifications to either the phosphate-sugar
backbone or the nucleoside, e.g., to reduce susceptibility to cellular
nucleases, improve bioavailability, improve formulation characteristics,
and/or change other pharmacokinetic properties. For example, the
phosphodiester linkages of natural RNA may be modified to include at
least one nitrogen or sulfur heteroatom. Modifications in RNA structure
may be tailored to allow specific genetic inhibition while avoiding a
general response to dsRNA. Likewise, bases may be modified to block the
activity of adenosine deaminase. The RNAi construct may be produced
enzymatically or by partial/total organic synthesis, any modified
ribonucleotide can be introduced by in vitro enzymatic or organic
synthesis. Methods of chemically modifying RNA molecules can be adapted
for modifying RNAi constructs (see, e.g., Heidenreich et al. (1997)
Nucleic Acids Res, 25:776-780; Wilson et al. (1994) J Mol Recog 7:89-98;
Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al.
(1997) Antisense Nucleic Acid Drug Dev 7:55-61). Merely to illustrate,
the backbone of an RNAi construct can be modified with phosphorothioates,
phosphoramidate, phosphodithioates, chimeric
methylphosphonate-phosphodiesters, peptide nucleic acids,
5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g.,
2'-substituted ribonucleosides, a-configuration).

[0064]Alternatively, the RNAi construct is in the form of a hairpin
structure (named as hairpin RNA). The hairpin RNAs can be synthesized
exogenously or can be formed by transcribing from RNA polymerase III
promoters in vivo. Examples of making and using such hairpin RNAs for
gene silencing in mammalian cells are described in, for example, Paddison
et al., Genes Dev, 2002, 16:948-58; McCaffrey et al., Nature, 2002,
418:38-9; McManus et al., RNA, 2002, 8:842-50; Yu et al., Proc Natl Acad
Sci U S A, 2002, 99:6047-52). Preferably, such hairpin RNAs are
engineered in cells or in an animal to ensure continuous and stable
suppression of a desired gene. It is known in the art that siRNAs can be
produced by processing a hairpin RNA in the cell.

[0065]PCT application WO 01/77350 describes an exemplary vector for
bi-directional transcription of a transgene to yield both sense and
antisense RNA transcripts of the same transgene in a eukaryotic cell.
Accordingly, in certain embodiments, the present invention provides a
recombinant vector having the following unique characteristics: it
comprises a viral replicon having two overlapping transcription units
arranged in an opposing orientation and flanking a transgene for an RNAi
construct of interest, wherein the two overlapping transcription units
yield both sense and antisense RNA transcripts from the same transgene
fragment in a host cell.

[0066]In another embodiment, the invention relates to the use of ribozyme
molecules designed to catalytically cleave an mRNA transcripts to prevent
translation of mRNA (see, e.g., PCT International Publication WO90/11364,
published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225; and
U.S. Pat. No. 5,093,246). While ribozymes that cleave mRNA at
site-specific recognition sequences can be used to destroy particular
mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes
cleave mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement is
that the target mRNA has the following sequence of two bases: 5'-UG-3'.
The construction and production of hammerhead ribozymes is well known in
the art and is described more fully in Haseloff and Gerlach, 1988,
Nature, 334:585-591. The ribozymes of the present invention also include
RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one
which occurs naturally in Tetrahymena thermophila (known as the IVS or
L-19 IVS RNA) and which has been extensively described (see, e.g., Zaug,
et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science,
231:470-475; Zaug, et al., 1986, Nature, 324:429-433; published
International patent application No. WO88/04300 by University Patents
Inc.; Been and Cech, 1986, Cell, 47:207-216).

[0067]In a further embodiment, the invention relates to the use of DNA
enzymes to inhibit expression of one or more of the biomarker gene (e.g.,
hTERT, IGFBP-3, transferrin receptor or beta-catenin). DNA enzymes
incorporate some of the mechanistic features of both antisense and
ribozyme technologies. DNA enzymes are designed so that they recognize a
particular target nucleic acid sequence, much like an antisense
oligonucleotide, however much like a ribozyme they are catalytic and
specifically cleave the target nucleic acid. Briefly, to design an ideal
DNA enzyme that specifically recognizes and cleaves a target nucleic
acid, one of skill in the art must first identify the unique target
sequence. Preferably, the unique or substantially sequence is a G/C rich
of approximately 18 to 22 nucleotides. High G/C content helps insure a
stronger interaction between the DNA enzyme and the target sequence. When
synthesizing the DNA enzyme, the specific antisense recognition sequence
that will target the enzyme to the message is divided so that it
comprises the two arms of the DNA enzyme, and the DNA enzyme loop is
placed between the two specific arms. Methods of making and administering
DNA enzymes can be found, for example, in U.S. Pat. No. 6,110,462.

[0068]In certain aspects, the therapeutic agents of the present invention
include a small molecule (e.g., a peptidomimetic). Examples of small
molecules include, but are not limited to, small peptides or peptide-like
molecules (e.g., a peptidomimetic). As used herein, the term
"peptidomimetic" includes chemically modified peptides and peptide-like
molecules that contain non-naturally occurring amino acids, peptoids, and
the like. Peptidomimetics provide various advantages over a peptide,
including enhanced stability when administered to a subject. Methods for
identifying a peptidomimetic are well known in the art and include the
screening of databases that contain libraries of potential
peptidomimetics. For example, the Cambridge Structural Database contains
a collection of greater than 300,000 compounds that have known crystal
structures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)).
Where no crystal structure of a target molecule is available, a structure
can be generated using, for example, the program CONCORD (Rusinko et al.,
J. Chem. Inf. Comput. Sci. 29:251 (1989)). Another database, the
Available Chemicals Directory (Molecular Design Limited, Informations
Systems; San Leandro Calif.), contains about 100,000 compounds that are
commercially available and also can be searched to identify potential
peptidomimetics.

[0069]As described herein, small molecule compounds may encompass numerous
chemical classes, though typically they are organic molecules, preferably
small organic compounds having a molecular weight of more than 50 and
less than about 2,500 daltons. Candidate agents comprise functional
groups necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine, carbonyl,
hydroxyl, sulfhydryl or carboxyl group. Candidate small molecule
compounds can be obtained from a wide variety of sources including
libraries of synthetic or natural compounds. For example, numerous means
are available for random and directed synthesis of a wide variety of
organic compounds and biomolecules, including expression of randomized
oligonucleotides. Alternatively, libraries of natural compounds in the
form of bacterial, fungal, plant, and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds can be modified through conventional chemical,
physical, and biochemical means. Known pharmacological agents may be
subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, and amidification, to produce
structural analogs.

[0070]In certain embodiments, methods of the present invention comprise
administering a therapeutically effective amount of a therapeutic agent
as described above. The phrase "therapeutically effective amount," as
used herein, refers to an amount that kills or inhibits (partially or
completely) growth of cervical cancer cells (e.g., HPV infected cells).
The dose of a therapeutic agent administered to an individual in need of
treatment will vary and will be determined for each individual with
reference to, for example, the compound used, the route of
administration, and the physical condition and body size of the
individual. The daily dosage may be administered as a single dosage or
may be divided into multiple doses. Actual dosage levels of a therapeutic
agent may be varied so as to obtain amounts at the site of target cells
(e.g., cervical cancer cells), effective to obtain the desired
therapeutic or prophylactic response.

[0071]In certain embodiments, the subject methods of the invention can be
used alone. Alternatively, the subject methods may be used in combination
with other anti-viral or anti-cancer therapeutic approaches (e.g.,
administration of an anti-viral or anti-cancer agent, radiation therapy,
phototherapy or immunotherapy) directed to treatment or prevention of
cervical cancer or virus infections. For example, such methods can be
used in prophylactic cancer prevention, prevention of cancer recurrence
and metastases after surgery, and as an adjuvant of other traditional
cancer therapy. Similarly, the subject methods of the invention may be
combined with other antiviral therapies.

[0072]Thus, the subject methods of the invention may further include as
optional ingredients one or more agents already known for their use in
the inhibition of cervical cancer, for added clinical efficacy. These
agents include, but are not limited to, interleukin-2, 5'-fluorouracil,
nedaplatin, methotrexate, vinblastine, doxorubicin, carboplatin,
paclitaxel (Taxol), cisplatin, 13-cis retinoic acid, pyrazoloacridine,
vinorelbine, artemisinin, and artemisinin analogs. Appropriate amounts in
each case will vary with the particular agent, and will be either readily
known to those skilled in the art or readily determinable by routine
experimentation. In other cases, the subject methods of the invention may
further include as optional ingredients one or more agents already known
for their anti-viral effects, for added clinical efficacy. These agents
include, but are not limited to, 5'-fluorouracil, interferon alpha,
imiquimod, landvudine, arsenic trioxide, capsaicin, nucleoside analogues
(e.g., acyclovir), and antiviral vaccines.

[0073]The present invention also contemplates therapeutic agents
obtainable from the screening methods described as below.

Drug Screening Assays

[0074]There are numerous approaches to screening for therapeutic agents in
cervical cancer therapy, which target one or more of the biomarkers
(e.g., hTERT, IGFBP-3, transferrin receptor, beta-catenin or Myc-HPV E6
interaction). For example, high-throughput screening of compounds or
molecules can be carried out to identify agents or drugs which inhibit
cervical cancer. Test agents to be assessed can be any chemical (element,
molecule, compound, drug), made synthetically, made by recombinant
techniques or isolated from a natural source. For example, test agents
can be peptides, polypeptides, peptoids, sugars, hormones, or nucleic
acid molecules (such as antisense or RNAi nucleic acid molecules). In
addition, test agents can be small molecules or molecules of greater
complexity made by combinatorial chemistry, for example, and compiled
into libraries. These libraries can comprise, for example, alcohols,
alkyl halides, amines, amides, esters, aldehydes, ethers and other
classes of organic compounds. Test agents can also be natural or
genetically engineered products isolated from lysates or growth media of
cells (e.g., bacterial, animal or plant), or can be the cell lysates or
growth media themselves. Presentation of test compounds to the test
system can be in either an isolated form or as mixtures of compounds,
especially in initial screening steps.

[0075]In one embodiment, the present invention provides assays to screen
for compounds that specifically inhibit protein-protein interaction
(e.g., binding of Myc to HPV E6). To illustrate, such compounds can be
identified by inhibition of binding of labeled Myc to HPV E6-Fc fusion
protein. Compounds identified through this screening can then be tested
in animal models of cervical cancer to assess their anti-tumor activity
in vivo. An assay to identify a substance which interferes with
interaction between Myc and HPV E6 can be performed with the component
(e.g., cells, purified protein, including fusion proteins and portions
having binding activity) which is not to be in competition with a test
compound, linked to a solid support. The solid support can be any
suitable solid phase or matrix, such as a bead, the wall of a plate or
other suitable surface (e.g., a well of a microtiter plate), column pore
glass (CPG) or a pin that can be submerged into a solution, such as in a
well. Linkage of cells or purified protein to the solid support can be
either direct or through one or more linker molecules.

[0076]In certain cases of the assays, an isolated or purified protein
(e.g., a Myc polypeptide) can be immobilized on a suitable affinity
matrix by standard techniques, such as chemical cross-linking, or via an
antibody raised against the isolated or purified protein, and bound to a
solid support. The matrix can be packed in a column or other suitable
container and is contacted with one or more compounds (e.g., a mixture)
to be tested under conditions suitable for binding of the compound to the
protein. For example, a solution containing compounds can be made to flow
through the matrix. The matrix can be washed with a suitable wash buffer
to remove unbound compounds and non-specifically bound compounds.
Compounds which remain bound can be released by a suitable elution
buffer. For example, a change in the ionic strength or pH of the elution
buffer can lead to a release of compounds. Alternatively, the elution
buffer can comprise a release component or components designed to disrupt
binding of compounds (e.g., one or more ligands or receptors, as
appropriate, or analogs thereof which can disrupt binding or
competitively inhibit binding of test compound to the protein).

[0077]In other embodiments, the present invention provides assays for
screening for compounds that decrease or block the expression level
(protein or nucleic acid) of a biomarker (hTERT, IGFBP-3, transferrin
receptor or beta-catenin). Methods of detecting and optionally
quantitating proteins can be achieved by techniques such as
antibody-based detection assays. In these cases, antibodies may be used
in a variety of detection techniques, including enzyme-linked
immunosorbent assays (ELISAs), immunoprecipitations, and Western blots.

[0078]On the other hand, methods of detecting and optionally quantitating
nucleic acids generally involve preparing purified nucleic acids and
subjecting the nucleic acids to a direct detection assay or an
amplification process followed by a detection assay. Amplification may be
achieved, for example, by polymerase chain reaction (PCR), reverse
transcriptase (RT), and coupled RT-PCR. Detection of nucleic acids is
generally accomplished by probing the purified nucleic acids with a probe
that hybridizes to the nucleic acids of interest, and in many instances,
detection involves an amplification step as well. Northern blots, dot
blots, microarrays, quantitative PCR, and quantitative RT-PCR are all
well known methods for detecting nucleic acids.

[0080]In some cases, one or more compounds can be tested simultaneously.
Where a mixture of compounds is tested, the compounds selected by the
foregoing processes can be separated (as appropriate) and identified by
suitable methods (e.g., PCR, sequencing, chromatography). Large
combinatorial libraries of compounds (e.g., organic compounds, peptides,
nucleic acids) produced by combinatorial chemical synthesis or other
methods can be tested (see e.g., Ohlmeyer, M. H. J. et al., Proc. Natl.
Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H. et al., Proc.
Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to tagged compounds;
see also, Rutter, W. J. et al., U.S. Pat. No. 5,010,175; Huebner, V. D.
et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No.
4,833,092). Where compounds selected from a combinatorial library by the
present method carry unique tags, identification of individual compounds
by chromatographic methods is possible. Where compounds do not carry
tags, chromatographic separation, followed by mass spectrophotometry to
ascertain structure, can be used to identify individual compounds
selected by the method, for example.

Pharmaceutical Compositions

[0081]In certain embodiments, therapeutic agents (compounds) of the
present invention are formulated with a pharmaceutically acceptable
carrier. Therapeutic agents of the present invention can be administered
alone or as a component of a pharmaceutical formulation (composition).
The compounds may be formulated for administration in any convenient way
for use in human medicine. Wetting agents, emulsifiers and lubricants,
such as sodium lauryl sulfate and magnesium stearate, as well as coloring
agents, release agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be present in
the compositions.

[0082]Formulations of the compounds include those suitable for oral/
nasal, topical, parenteral and/or intravaginal administration. The
formulations may conveniently be presented in unit dosage form and may be
prepared by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the individual
being treated and the particular mode of administration. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form can be an amount of the compound which
produces a therapeutic effect. Alternatively, multiple doses can be taken
by an individual.

[0083]Methods of preparing these formulations or compositions include
combining one or more compounds with one or more carriers and,
optionally, one or more accessory ingredients. For example, the
formulations are prepared by combining a compound with a liquid carrier,
or a finely divided solid carrier, or both, and then, if necessary,
shaping the product.

[0084]Formulations of the compounds suitable for oral administration may
be in the form of capsules, cachets, pills, tablets, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or non-aqueous
liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an
elixir or syrup, or as pastilles (using an inert base, such as gelatin
and glycerin, or sucrose and acacia) and/or as mouth washes and the like,
each containing a predetermined amount of a compound as an active
ingredient. A compound may also be administered as a bolus, electuary or
paste.

[0085]In solid dosage forms for oral administration (capsules, tablets,
pills, dragees, powders, granules, and the like), a compound is mixed
with one or more pharmaceutically acceptable carriers, such as sodium
citrate or dicalcium phosphate, and/or any of the following: (1) fillers
or extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose, and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato or
tapioca starch, alginic acid, certain silicates, and sodium carbonate;
(5) solution retarding agents, such as paraffin; (6) absorption
accelerators, such as quaternary ammonium compounds; (7) wetting agents,
such as, for example, cetyl alcohol and glycerol monostearate; (8)
absorbents, such as kaolin and bentonite clay; (9) lubricants, such a
talc, calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In
the case of capsules, tablets and pills, the pharmaceutical compositions
may also comprise buffering agents. Solid compositions of a similar type
may also be employed as fillers in soft and hard-filled gelatin capsules
using such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.

[0088]In particular, methods of the invention can be administered
topically, either to skin or to mucosal membranes such as those on the
cervix and vagina. This offers the greatest opportunity for direct
delivery to tumor with the lowest chance of inducing side effects. The
topical formulations may further include one or more of the wide variety
of agents known to be effective as skin or stratum corneum penetration
enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone,
dimethylacetamide, dimethylformamide, propylene glycol, methyl or
isopropyl alcohol, dimethyl sulfoxide, and atone. Additional agents may
further be included to make the formulation cosmetically acceptable.
Examples of these are fats, waxes, oils, dyes, fragrances, preservatives,
stabilizers, and surface active agents. Keratolytic agents such as those
known in the art may also be included. Examples are salicylic acid and
sulfur.

[0089]Dosage forms for the topical or transdermal administration of a
compound include powders, sprays, ointments, pastes, creams, lotions,
gels, solutions, patches, and inhalants. The active compound may be mixed
under sterile conditions with a pharmaceutically acceptable carrier, and
with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to a
therapeutic compound, excipients, such as animal and vegetable fats,
oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,
polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc
oxide, or mixtures thereof.

[0090]Powders and sprays can contain, in addition to a compound,
excipients such as lactose, talc, silicic acid, aluminum hydroxide,
calcium silicates, and polyamide powder, or mixtures of these substances.
Sprays can additionally contain customary propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as
butane and propane.

[0091]Pharmaceutical compositions suitable for parenteral administration
may comprise one or more compounds in combination with one or more
pharmaceutically acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile powders
which may be reconstituted into sterile injectable solutions or
dispersions just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with the
blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be
employed in the pharmaceutical compositions of the invention include
water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable oils,
such as olive oil, and injectable organic esters, such as ethyl oleate.
Proper fluidity can be maintained, for example, by the use of coating
materials, such as lecithin, by the maintenance of the required particle
size in the case of dispersions, and by the use of surfactants.

[0092]Injectable depot forms are made by forming microencapsule matrices
of the compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to polymer, and
the nature of the particular polymer employed, the rate of drug release
can be controlled. Examples of other biodegradable polymers include
poly(orthoesters) and poly(anhydrides). Depot injectable formulations are
also prepared by entrapping the drug in liposomes or microemulsions which
are compatible with body tissue.

[0093]Formulations of the compounds for intravaginal administration may be
presented as a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which is
solid at room temperature, but liquid at body temperature and, therefore,
will melt in the rectum or vaginal cavity and release the active
compound. Optionally, such formulations suitable for vaginal
administration also include pessaries, tampons, creams, gels, pastes,
foams or spray formulations containing such carriers as are known in the
art to be appropriate.

INCORPORATION BY REFERENCE

[0094]All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated to be
incorporated by reference.

[0095]While specific embodiments of the subject invention have been
discussed, the above specification is illustrative and not restrictive.
Many variations of the invention will become apparent to those skilled in
the art upon review of this specification and the claims below. The full
scope of the invention should be determined by reference to the claims,
along with their full scope of equivalents, and the specification, along
with such variations.